PROJECT SUMMARY In response to emergent resistance to other artemisinin-based combination therapies, dihydroartemisinin- piperaquine became the drug of choice to treat artemisinin-resistant Plasmodium falciparum in Southeast Asia and is being used in large scale targeted mass treatment field evaluations to eliminate malaria in the region in hopes of preventing the spread of drug resistance. However, the emergence of piperaquine resistance is compromising the last effective antimalarial for Southeast Asia, which bodes poorly for the ability to treat this important disease in this part of the world. Identification of molecular markers for use in surveillance would provide a rapid means to measure the present extent of piperaquine resistance and guide rational containment strategies to deter its further spread. We have conducted a genome-wide association study that has identified multiple regions of the parasite genome that are significantly associated with ex vivo piperaquine susceptibility. We propose to identify specific genes and polymorphisms within these regions of the P. falciparum genome and to validate these candidate resistance markers in the field and in the laboratory. The work will be accomplished in three aims. First, we will identify candidate piperaquine resistance genes and polymorphisms using an annotated reference P. falciparum genome assembly for Southeast Asia. Candidate resistance genes will be identified and prioritized, and whole genome sequencing will be used to identify single nucleotide polymorphisms within candidate genes or gene copy number variants and their association with ex vivo piperaquine susceptibility. Rapid assays will be developed to genotype candidate resistance markers significantly associated with the phenotype. Second, we will use the rapid assays developed as part of Aim 1 to genotype candidate piperaquine resistance markers in P. falciparum infections collected as part of dihydroartemisinin-piperaquine efficacy studies, and we will estimate the association between candidate resistance markers and clinical outcomes following treatment with dihydroartemisinin-piperaquine. Third, we will use CRISPR/Cas9-mediated gene editing to introduce candidate resistance mutations identified in Aims 1 and 2 into piperaquine-sensitive parasite isolates and to remove candidate resistance mutations from piperaquine-resistant parasite isolates to validate the role of these mutations in determining susceptibility to piperaquine. If successful, this project will result in a greater understanding of the molecular mechanisms underlying piperaquine resistance, as well as genetic markers that can be used to track and contain piperaquine-resistant parasites and guide malaria drug treatment policies.